Certain plant pests particularly Colepteran insects and those insects of the genus Diabrotica cause tremendous damage to crop plants. In the past and currently, chemical fumigation has been used to control such insect damage. Additionally crop rotation practices can control Diabrotica populations. However, excessive use of insecticidal chemicals is not environmentally desirable and the practice of crop rotation may not be feasible for all growers. There is also increasing evidence that annual crop rotation may not provide complete protection from Diabrotica due to the increased prevalence of extended diapause in some Diabrotica populations. Therefore a means of protecting the plants from such insect damage by a mechanism other than chemical control or crop rotation would be very beneficial.
Proteases or peptidases, hereinafter referred to collectively as proteases, are enzymes which hydrolyze peptide bonds in proteins or peptides and demonstrate this activity at the ends of peptides (exopeptidases) or within the peptide chain (endopeptidases). The endopeptidases in particular cleave internal peptide bonds with different degrees of specificity for particular amino acyl residues. The protease enzymes are classified on the basis of their catalytic residues. Thus, with thiol proteases a cysteine sulfhydryl group participates directly in cleavage of the substrate peptide bond. Proteases are ubiquitous in nature and it is well documented that these enzymes are present in insect and other plant pests.
Peptides are generated from proteins in the gastrointestinal tract of organisms by the action of proteases during digestion. These organisms must rely on the activity of a battery of potent gut proteases in order to obtain all amino acids. The best characterized insect proteases are serine proteases (Applebaum, S. W. (1985) Biochemistry of Digestion, in Comprehensive Insect Physiology. Biochemistry and Pharmacology, Kerkut, G. A. and Gilbert, L. eds. 4:279-311. Pergamon Press, Oxford). In contrast, less polyphagous insects may employ acid proteases (Pendola, S. and B. Greenburg, (1975) Ann. Ent. Soc. Am. 69:341-345) or thiol proteases (Murdock et at, (1987), Comp. Biochem. Physiol. 87B:783-787) as major contributors in protein digestion. The gut of Diabrotica virgifera, common name, western corn rootworm (wCRW) has been found to be particularly rich in thiol proteases (Murdock et al, supra).
Molecules which form complexes with proteases and inhibit their proteolytic activity are also widespread in nature and they are regulators of proteolytic activity. The presence of a peptide inhibitor of thiol proteases, known as cystatin, was first described in 1957. Since this initial discovery, many cystatin type inhibitors have been characterized, and these inhibitors are generally referred to as members of the cystatin superfamily. The three subgroups of the superfamily include: the cystatins, containing disulfide bond(s); the stefins, lacking cysteine residues; and the kininogens which are high molecular weight glycoproteins with disulfide bonds.
Protease inhibitors are among the defensive chemicals in plant tissue that are both developmentally regulated and induced in response to insect and/or pathogen attack. Suppression of serine protease inhibitor expression in transgenic tomato plants has been demonstrated to result in reduced tolerance to insect feeding.
Certain thiol protease inhibitors that are toxic to insects and in particular Coleoptera are well characterized. In three species of beetles studied by Murdock et al, (supra) it was found that much or most of the proteolytic activity in the midgut extracts was inhibited by E-64, [N-(L-3 transcarrboxyoxiran-2-carbonyl)-L-leucyl]-amido(4-guanido)-butane, a specific, potent nonprotein inhibitor of thiol proteases.
The present invention is particularly concerned with the discovery of a novel peptide designated virgiferin as an effective thiol protease inhibitor. The amino acid sequence of virgiferin and modified virgiferin distinguishes these peptides from thiol protease inhibitor members of the cystatin superfamily.